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Articles by Philippe Bastin
In JoVE (1)
Other Publications (51)
- Molecular and Biochemical Parasitology
- The EMBO Journal
- BMC Biology
- Journal De La Societe De Biologie
- BMC Biotechnology
- International Review of Cytology
- Journal of Cell Science
- PloS One
- Molecular and Biochemical Parasitology
- Molecular Biology of the Cell
- Journal of Cell Science
- Cellular Microbiology
- Journal of Cell Science
- American Journal of Human Genetics
- The Journal of Cell Biology
- Methods in Cell Biology
- Journal of Cell Science
- Current Opinion in Microbiology
- PLoS Neglected Tropical Diseases
- Cellular Microbiology
- Biology of the Cell
- Biology of the Cell
- Cellular Microbiology
- Biologie Aujourd'hui
- Molecular Biology of the Cell
- PLoS Biology
- Development (Cambridge, England)
- Journal of Cell Science
- Eukaryotic Cell
- Antimicrobial Agents and Chemotherapy
- Science (New York, N.Y.)
- Cilia
- Frontiers in Cellular and Infection Microbiology
- Cellular Microbiology
- Journal of Cell Science
- ELife
- Molecular & Cellular Proteomics : MCP
- Molecular Microbiology
- ELife
- Molecular Biology of the Cell
- Medecine Sciences : M/S
- PloS One
- Trends in Parasitology
- Methods in Cell Biology
- Cilia
- Micron (Oxford, England : 1993)
- PloS One
- Journal of Medical Genetics
- Biology Open
- Journal of Cell Science
- JAMA Oncology
Articles by Philippe Bastin in JoVE
Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography Sylvain Trépout1, Philippe Bastin2, Sergio Marco1 1Institut Curie, INSERM U1196, Campus Universitaire d'Orsay, 2Institut Pasteur, Trypanosome Cell Biology Unit, Department of Parasites & Insect Vectors, INSERM U1201 This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.
Other articles by Philippe Bastin on PubMed
Efficiency and Specificity of RNA Interference Generated by Intra- and Intermolecular Double Stranded RNA in Trypanosoma Brucei Molecular and Biochemical Parasitology. Jun, 2003 | Pubmed ID: 12798502 In many eukaryotes, double-stranded (ds) RNA leads to specific degradation of RNA of cognate sequence, a process termed RNA interference (RNAi). Here we used the protozoan Trypanosoma brucei as a model to investigate efficiency and specificity of RNAi generated by expression of long dsRNA of PFRA and PFRC genes, which code for flagellar proteins required for cell motility. Consequences of RNAi were monitored at all three levels: target RNA expression, protein expression and phenotype observation, using population or individual cell analysis. Expression of PFRA dsRNA from an inverted repeat was extremely efficient, knocking down PFRA RNA and PFRA protein, and producing a severe paralysis phenotype. Silencing by expression of PFRA dsRNA using a dual facing promoter system was also very efficient, producing a clear phenotype, although low amounts of PFRA RNA and PFRA protein were detected. Expression via the dual facing promoters of PAR2 dsRNA (83% overall identity with PFRA, including nine blocks of >20 nt total identity) did not produce significant reduction of total amounts of PFRA RNA or PFRA protein. However, individual cell analysis by immunofluorescence revealed that 10-60% cells (depending on subclones) exhibited lower PFRA amounts in their flagellum, producing a reduced-motility phenotype.
Novel Roles for the Flagellum in Cell Morphogenesis and Cytokinesis of Trypanosomes The EMBO Journal. Oct, 2003 | Pubmed ID: 14532107 Flagella and cilia are elaborate cytoskeletal structures conserved from protists to mammals, where they fulfil functions related to motility or sensitivity. Here we demonstrate novel roles for the flagellum in the control of cell size, shape, polarity and division of the protozoan Trypanosoma brucei. To investigate the function of the flagellum, its formation was perturbed by inducible RNA interference silencing of com ponents required for intraflagellar transport, a dynamic process necessary for flagellum assembly. First, we show that down-regulation of intraflagellar transport leads to assembly of a shorter flagellum. Strikingly, cells with a shorter flagellum are smaller, with a direct correlation between flagellum length and cell size. Detailed morphogenetic analysis reveals that the tip of the new flagellum defines the point where cytokinesis is initiated. Secondly, when new flagellum formation is completely blocked, non-flagellated cells are very short, lose their normal shape and polarity, and fail to undergo cytokinesis. We show that flagellum elongation controls formation of cytoskeletal structures (present in the cell body) that act as molecular organizers of the cell.
TbAGO1, an Argonaute Protein Required for RNA Interference, is Involved in Mitosis and Chromosome Segregation in Trypanosoma Brucei BMC Biology. Dec, 2003 | Pubmed ID: 14670085 RNA silencing processes are widespread in almost all eukaryotic organisms. They have various functions including genome protection, and the control of gene expression, development and heterochromatin formation. RNA interference (RNAi) is the post-transcriptional destruction of RNA, which is mediated by a ribonucleoprotein complex that contains, among several components, RNA helicases and Argonaute proteins. RNAi is functional in trypanosomes, protozoan parasites that separated very early from the main eukaryotic lineage and exhibit several intriguing features in terms of the control of gene expression. In this report, we investigated the functions of RNAi in Trypanosoma brucei.
[The Flagellum: from Cell Motility to Morphogenesis] Journal De La Societe De Biologie. 2003 | Pubmed ID: 15005520 Flagella and cilia are elaborate cytoskeletal structures conserved from protists to mammals, where they fulfil functions related to motility or sensitivity. We demonstrate a novel role for the flagellum in the control of cell morphogenesis and division of Trypanosoma brucei. To investigate flagellum functions, its formation was perturbed by inducible RNA interference silencing of components required for intraflagellar transport (IFT), a dynamic process necessary for flagellum assembly. First, we show that down-regulation of IFT leads to assembly of a shorter flagellum. Strikingly, cells with a shorter flagellum are smaller, with a direct correlation between flagellum length and cell size. Detailed morphogenetic analysis reveals that the tip of the new flagellum defines the point where cytokinesis is initiated. Furthermore, when new flagellum formation is completely blocked, non-flagellated cells are very short, lose their normal shape and polarity and fail to undergo cytokinesis. We show that flagellum elongation controls formation of cytoskeletal structures present in the cell body that act as molecular organisers of the cell.
Functional Complementation of RNA Interference Mutants in Trypanosomes BMC Biotechnology. Feb, 2005 | Pubmed ID: 15703078 In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments.
The Flagellum of Trypanosomes International Review of Cytology. 2005 | Pubmed ID: 16157182 Eukaryotic cilia and flagella are cytoskeletal organelles that are remarkably conserved from protists to mammals. Their basic unit is the axoneme, a well-defined cylindrical structure composed of microtubules and up to 250 associated proteins. These complex organelles are assembled by a dynamic process called intraflagellar transport. Flagella and cilia perform diverse motility and sensitivity functions in many different organisms. Trypanosomes are flagellated protozoa, responsible for various tropical diseases such as sleeping sickness and Chagas disease. In this review, we first describe general knowledge on the flagellum: its occurrence in the living world, its molecular composition, and its mode of assembly, with special emphasis on the exciting developments that followed the discovery of intraflagellar transport. We then present recent progress regarding the characteristics of the trypanosome flagellum, highlighting the original contributions brought by this organism. The most striking phenomenon is the involvement of the flagellum in several aspects of the trypanosome cell cycle, including cell morphogenesis, basal body migration, and cytokinesis.
Conserved and Specific Functions of Axoneme Components in Trypanosome Motility Journal of Cell Science. Aug, 2006 | Pubmed ID: 16882690 The Trypanosoma brucei flagellum is unusual as it is attached along the cell body and contains, in addition to an apparently conventional axoneme, a structure called the paraflagellar rod, which is essential for cell motility. Here, we investigated flagellum behaviour in normal and mutant trypanosome cell lines where expression of genes encoding various axoneme proteins (PF16, PF20, DNAI1, LC2) had been silenced by RNAi. First, we show that the propulsive wave (normally used for forward motility) is abolished in the absence of outer dynein arms, whereas the reverse wave (normally used for changing direction) still occurs. Second, in contrast to Chlamydomonas--but like metazoa, the central pair adopts a fixed orientation during flagellum beating. This orientation becomes highly variable in central-pair- and outer-dynein-arm-mutants. Third, the paraflagellar rod contributes to motility by facilitating three-dimensional wave propagation and controlling cell shape. Fourth, motility is required to complete the last stage of cell division in both insect and bloodstream stages of the parasite. Finally, our study also reveals the conservation of molecular components of the trypanosome flagellum. Coupled to the ease of reverse genetics, it raises the interest of trypanosomes as model organisms to study cilia and flagella.
Basal Body Positioning is Controlled by Flagellum Formation in Trypanosoma Brucei PloS One. May, 2007 | Pubmed ID: 17487282 To perform their multiple functions, cilia and flagella are precisely positioned at the cell surface by mechanisms that remain poorly understood. The protist Trypanosoma brucei possesses a single flagellum that adheres to the cell body where a specific cytoskeletal structure is localised, the flagellum attachment zone (FAZ). Trypanosomes build a new flagellum whose distal tip is connected to the side of the old flagellum by a discrete structure, the flagella connector. During this process, the basal body of the new flagellum migrates towards the posterior end of the cell. We show that separate inhibition of flagellum assembly, base-to-tip motility or flagella connection leads to reduced basal body migration, demonstrating that the flagellum contributes to its own positioning. We propose a model where pressure applied by movements of the growing new flagellum on the flagella connector leads to a reacting force that in turn contributes to migration of the basal body at the proximal end of the flagellum.
The Argonaute Protein TbAGO1 Contributes to Large and Mini-chromosome Segregation and is Required for Control of RIME Retroposons and RHS Pseudogene-associated Transcripts Molecular and Biochemical Parasitology. Dec, 2007 | Pubmed ID: 17822785 The protist Trypanosoma brucei possesses a single Argonaute gene called TbAGO1 that is necessary for RNAi silencing. We previously showed that in strain 427, TbAGO1 knock-out leads to a slow growth phenotype and to chromosome segregation defects. Here we report that the slow growth phenotype is linked to defects in segregation of both large and mini-chromosome populations, with large chromosomes being the most affected. These phenotypes are completely reversed upon inducible re-expression of TbAGO1 fused to GFP, demonstrating their link with TbAGO1. Trypanosomes that do not express TbAGO1 show a general increase in the abundance of transcripts derived from the short retroposon RIME (Ribosomal Interspersed Mobile Element). Supplementary large RIME transcripts emerge in the absence of RNAi, a phenomenon coupled to the disappearance of short transcripts. These fluctuations are reversed by inducible expression of GFP::TbAGO1. Furthermore, we use a combination of Northern blots, RT-PCR and sequencing to reveal that RNAi controls expression of transcripts derived from RHS (Retrotransposon Hot Spot) pseudogenes (RHS genes with retro-element(s) integrated within their coding sequence). Absence of RNAi also leads to an increase of steady-state transcripts from regular RHS genes (those without retro-element), indicating a role for pseudogene in control of gene expression. However, analysis of retroposon abundance and arrangement in the genome of multiple clonal cell lines of TbAGO1-/- failed to reveal movement of mobile elements despite the increased amounts of retroposon transcripts.
Intraflagellar Transport and Functional Analysis of Genes Required for Flagellum Formation in Trypanosomes Molecular Biology of the Cell. Mar, 2008 | Pubmed ID: 18094047 Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3-4 and 7-8, suggesting the existence of specific IFT tracks. Rapid (>3 microm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant.
Flagellum Elongation is Required for Correct Structure, Orientation and Function of the Flagellar Pocket in Trypanosoma Brucei Journal of Cell Science. Nov, 2008 | Pubmed ID: 18940910 In trypanosomes, the flagellum is rooted in the flagellar pocket, a surface micro-domain that is the sole site for endocytosis and exocytosis. By analysis of anterograde or retrograde intraflagellar transport in IFT88RNAi or IFT140RNAi mutant cells, we show that elongation of the new flagellum is not required for flagellar pocket formation but is essential for its organisation, orientation and function. Transmission electron microscopy revealed that the flagellar pocket exhibited a modified shape (smaller, distorted and/or deeper) in cells with abnormally short or no flagella. Scanning electron microscopy analysis of intact and detergent-extracted cells demonstrated that the orientation of the flagellar pocket collar was more variable in trypanosomes with short flagella. The structural protein BILBO1 was present but its localisation and abundance was altered. The membrane flagellar pocket protein CRAM leaked out of the pocket and reached the short flagella. CRAM also accumulated in intracellular compartments, indicating defects in routing of resident flagellar pocket proteins. Perturbations of vesicular trafficking were obvious; vesicles were observed in the lumen of the flagellar pocket or in the short flagella, and fluid-phase endocytosis was drastically diminished in non-flagellated cells. We propose a model to explain the role of flagellum elongation in correct flagellar pocket organisation and function.
The Flagellum-mitogen-activated Protein Kinase Connection in Trypanosomatids: a Key Sensory Role in Parasite Signalling and Development? Cellular Microbiology. May, 2009 | Pubmed ID: 19207727 Trypanosomatid parasites are the causative agents of severe human diseases such as sleeping sickness, Chagas disease and leishmaniases. These microorganisms are transmitted via different insect vectors and hence are confronted to changing environments during their infectious cycle in which they activate specific and complex patterns of differentiation. Several studies in Trypanosoma brucei and in different subspecies of Leishmania have shed light on the role of mitogen-activated protein (MAP) kinases in these processes. Surprisingly, several MAP kinases turned out to be involved in the control of flagellum length in the promastigote stage of Leishmania. Recently, a sensory function has been recognized for cilia and flagella in unicellular and multicellular eukaryotes. This review aims to stimulate discussions on the possibility that the Trypanosomatid flagellum could act as a sensory organ through the MAP kinase pathway, with the objective to encourage investigation of this new hypothesis through a series of proposed experimental approaches.
A Novel Function for the Atypical Small G Protein Rab-like 5 in the Assembly of the Trypanosome Flagellum Journal of Cell Science. Mar, 2009 | Pubmed ID: 19240117 The atypical small G protein Rab-like 5 has been shown to traffic in sensory cilia of Caenorhabditis elegans, where it participates in signalling processes but not in cilia construction. In this report, we demonstrate that RABL5 colocalises with intraflagellar transport (IFT) proteins at the basal body and in the flagellum matrix of the protist Trypanosoma brucei. RABL5 fused to GFP exhibits anterograde movement in the flagellum of live trypanosomes, suggesting it could be associated with IFT. Accordingly, RABL5 accumulates in the short flagella of the retrograde IFT140(RNAi) mutant and is restricted to the basal body region in the IFT88(RNAi) anterograde mutant, a behaviour that is identical to other IFT proteins. Strikingly, RNAi silencing reveals an essential role for RABL5 in trypanosome flagellum construction. RNAi knock-down produces a phenotype similar to inactivation of retrograde IFT with formation of short flagella that are filled with a high amount of IFT proteins. These data reveal for the first time a functional difference for a conserved flagellar matrix protein between two different ciliated species and raise questions related to cilia diversity.
Loss-of-function Mutations in the Human Ortholog of Chlamydomonas Reinhardtii ODA7 Disrupt Dynein Arm Assembly and Cause Primary Ciliary Dyskinesia American Journal of Human Genetics. Dec, 2009 | Pubmed ID: 19944405 Cilia and flagella are evolutionarily conserved structures that play various physiological roles in diverse cell types. Defects in motile cilia result in primary ciliary dyskinesia (PCD), the most prominent ciliopathy, characterized by the association of respiratory symptoms, male infertility, and, in nearly 50% of cases, situs inversus. So far, most identified disease-causing mutations involve genes encoding various ciliary components, such those belonging to the dynein arms that are essential for ciliary motion. Following a candidate-gene approach based on data from a mutant strain of the biflagellated alga Chlamydomonas reinhardtii carrying an ODA7 defect, we identified four families with a PCD phenotype characterized by the absence of both dynein arms and loss-of-function mutations in the human orthologous gene called LRRC50. Functional analyses performed in Chlamydomonas reinhardtii and in another flagellated protist, Trypanosoma brucei, support a key role for LRRC50, a member of the leucine-rich-repeat superfamily, in cytoplasmic preassembly of dynein arms.
Kinesin 9 Family Members Perform Separate Functions in the Trypanosome Flagellum The Journal of Cell Biology. Nov, 2009 | Pubmed ID: 19948486 Numerous eukaryote genome projects have uncovered a variety of kinesins of unknown function. The kinesin 9 family is limited to flagellated species. Our phylogenetic experiments revealed two subfamilies: KIF9A (including Chlamydomonas reinhardtii KLP1) and KIF9B (including human KIF6). The function of KIF9A and KIF9B was investigated in the protist Trypanosoma brucei that possesses a single motile flagellum. KIF9A and KIF9B are strongly associated with the cytoskeleton and are required for motility. KIF9A is localized exclusively in the axoneme, and its depletion leads to altered motility without visible structural modifications. KIF9B is found in both the axoneme and the basal body, and is essential for the assembly of the paraflagellar rod (PFR), a large extra-axonemal structure. In the absence of KIF9B, cells grow abnormal flagella with excessively large blocks of PFR-like material that alternate with regions where only the axoneme is present. The functional diversity of the kinesin 9 family illustrates the capacity for adaptation of organisms to suit specific cytoskeletal requirements.
Tools for Analyzing Intraflagellar Transport in Trypanosomes Methods in Cell Biology. 2009 | Pubmed ID: 20409811 African trypanosomes are evolutionary-divergent eukaryotes responsible for sleeping sickness. They duplicate their single flagellum while maintaining the old one, providing a unique model to examine mature and elongating flagella in the same cell. Like in most eukaryotes, the trypanosome flagellum is constructed by addition of novel subunits at its distal end via the action of intraflagellar transport (IFT). Almost all genes encoding IFT proteins and motors are conserved in trypanosomes and related species, with only a few exceptions. A dozen of IFT genes have been functionally investigated in this organism, thanks to the potent reverse genetic tools available. Several alternative techniques to trigger RNAi are accessible, either transient RNAi by transfection of long double-stranded RNA or by generation of clonal cell lines able to express long double-stranded RNA under the control of tetracycline-inducible promoters. In addition, we provide a series of techniques to investigate cellular phenotypes in trypanosomes where expression of IFT genes has been silenced. In this chapter, we describe different methods for tagging and expression of IFT proteins in trypanosomes and for visualizing IFT in live cells.
The Ciliary Pocket: an Endocytic Membrane Domain at the Base of Primary and Motile Cilia Journal of Cell Science. May, 2010 | Pubmed ID: 20427320 Cilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actin-based cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia.
Cyclosporin A Treatment of Leishmania Donovani Reveals Stage-specific Functions of Cyclophilins in Parasite Proliferation and Viability PLoS Neglected Tropical Diseases. Jun, 2010 | Pubmed ID: 20614016 Cyclosporin A (CsA) has important anti-microbial activity against parasites of the genus Leishmania, suggesting CsA-binding cyclophilins (CyPs) as potential drug targets. However, no information is available on the genetic diversity of this important protein family, and the mechanisms underlying the cytotoxic effects of CsA on intracellular amastigotes are only poorly understood. Here, we performed a first genome-wide analysis of Leishmania CyPs and investigated the effects of CsA on host-free L. donovani amastigotes in order to elucidate the relevance of these parasite proteins for drug development.
Molecular Bases of Cytoskeleton Plasticity During the Trypanosoma Brucei Parasite Cycle Cellular Microbiology. May, 2011 | Pubmed ID: 21159115 African trypanosomes are flagellated protozoan parasites responsible for sleeping sickness and transmitted by tsetse flies. The accomplishment of their parasite cycle requires adaptation to highly diverse environments. These transitions take place in a strictly defined order and are accompanied by spectacular morphological modifications in cell size, shape and positioning of organelles. To understand the molecular bases of these processes, parasites isolated from different tissues of the tsetse fly were analysed by immunofluorescence with markers for specific cytoskeleton components and by a new immunofluorescence-based assay for evaluation of the cell volume. The data revealed striking differences between proliferative stages found in the midgut or in the salivary glands and the differentiating stage occurring in the proventriculus. Cell proliferation was characterized by a significant increase in cell volume, by a pronounced cell elongation marked by microtubule extension at the posterior end, and by the production of a new flagellum similar to the existing one. In contrast, the differentiating stage found in the proventriculus does not display any increase in cell volume neither in cell length, but is marked by a profound remodelling of the posterior part of the cytoskeleton and by changes in molecular composition and/or organization of the flagellum attachment zone.
1001 Model Organisms to Study Cilia and Flagella Biology of the Cell. Mar, 2011 | Pubmed ID: 21275904 Most mammalian cell types have the potential to assemble at least one cilium. Immotile cilia participate in numerous sensing processes, while motile cilia are involved in cell motility and movement of extracellular fluid. The functional importance of cilia and flagella is highlighted by the growing list of diseases due to cilia defects. These ciliopathies are marked by an amazing diversity of clinical manifestations and an often complex genetic aetiology. To understand these pathologies, a precise comprehension of the biology of cilia and flagella is required. These organelles are remarkably well conserved throughout eukaryotic evolution. In this review, we describe the strengths of various model organisms to decipher diverse aspects of cilia and flagella biology: molecular composition, mode of assembly, sensing and motility mechanisms and functions. Pioneering studies carried out in the green alga Chlamydomonas established the link between cilia and several genetic diseases. Moreover, multicellular organisms such as mouse, zebrafish, Xenopus, Caenorhabditis elegans or Drosophila, and protists such as Paramecium, Tetrahymena and Trypanosoma or Leishmania each bring specific advantages to the study of cilium biology. For example, the function of genes involved in primary ciliary dyskinesia (due to defects in ciliary motility) can be efficiently assessed in trypanosomes.
The Ciliary Pocket: a Once-forgotten Membrane Domain at the Base of Cilia Biology of the Cell. Mar, 2011 | Pubmed ID: 21275905 The PC (primary cilium) is present on most cell types in both developing and adult tissues in vertebrates. Despite multiple reports in the 1960s, the PC was almost forgotten for decades by most of the cell biology community, mainly because its function appeared enigmatic. This situation changed 10 years ago with the key discovery that this fascinating structure is the missing link between complex genetic diseases and key signalling pathways during development and tissue homoeostasis. A similar misfortune might have happened to an original membrane domain found at the base of PC in most cell types and recently termed the 'ciliary pocket'. A morphologically related structure has also been described at the connecting cilium of photoreceptors and at the flagellum in spermatids. Its organization is also reminiscent of the flagellar pocket, a plasma membrane invagination specialized in uptake and secretion encountered in kinetoplastid protozoa. The exact function of the ciliary pocket remains to be established, but the recent observation of endocytic activity coupled to the fact that vesicular trafficking plays important roles during ciliogenesis brought excitement in the ciliary community. Here, we have tried to decipher what this highly conserved membrane domain could tell us about the function and/or biogenesis of the associated cilium.
Quantitative Proteome Profiling Informs on Phenotypic Traits That Adapt Leishmania Donovani for Axenic and Intracellular Proliferation Cellular Microbiology. Jul, 2011 | Pubmed ID: 21501362 Protozoan parasites of the genus Leishmania are important human pathogens that differentiate inside host macrophages into an amastigote life cycle stage. Although this stage causes the pathogenesis of leishmaniasis, only few proteins have been implicated in amastigote intracellular survival. Here we compare morphology, infectivity and protein expression of L. donovani LD1S grown in host free (axenic) culture, or exclusively propagated in infected hamsters, with the aim to reveal parasite traits absent in axenic but selected for in hamster-derived amastigotes through leishmanicidal host activities. Axenic and splenic amastigotes showed a striking difference in virulence and the ability to cause experimental hepato-splenomegaly in infected hamsters. 2D-DIGE analysis revealed statistically significant differences in abundance for 152 spots, with 14 spots showing fivefold or higher abundance in splenic amastigotes. Proteins identified by MS analysis include the anti-oxidant enzyme tryparedoxin peroxidase, and enzymes implicated in protein and amino acid metabolism. Analysis of parasite growth in vitro in minimal medium demonstrated increased survival of hamster-derived compared with axenic parasites under conditions that mimic the nutrient poor, cytotoxic phagolysosome. Thus, our comparative proteomics analysis sheds important new light on the biochemistry of bona fide amastigotes and informs on survival factors relevant for intracellular L. donovani infection.
[The Importance of Model Organisms to Study Cilia and Flagella Biology] Biologie Aujourd'hui. 2011 | Pubmed ID: 21501571 Cilia and flagella are ubiquitous organelles that protrude from the surfaces of many cells, and whose architecture is highly conserved from protists to humans. These complex organelles, composed of over 500 proteins, can be either immotile or motile. They are involved in a myriad of biological processes, including sensing (non-motile cilia) and/or cell motility or movement of extracellular fluids (motile cilia). The ever-expanding list of human diseases linked to defective cilia illustrates the functional importance of cilia and flagella. These ciliopathies are characterised by an impressive diversity of symptoms and an often complex genetic etiology. A precise knowledge of cilia and flagella biology is thus critical to better understand these pathologies. However, multi-ciliated cells are terminally differentiated and difficult to manipulate, and a primary cilium is assembled only when the cell exits from the cell cycle. In this context the use of model organisms, that relies on the high degree of structural but also of molecular conservation of these organelles across evolution, is instrumental to decipher the many facets of cilia and flagella biology. In this review, we highlight the specific strengths of the main model organisms to investigate the molecular composition, mode of assembly, sensing and motility mechanisms and functions of cilia and flagella. Pioneering studies carried out in the green alga Chlamydomonas established the link between cilia and several genetic diseases. Moreover, multicellular organisms such as mouse, zebrafish, Xenopus, C. elegans or Drosophila, and protists like Paramecium, Tetrahymena and Trypanosoma or Leishmania each bring specific advantages to the study of cilium biology. For example, the function of genes involved in primary ciliary dyskinesia (due to defects in ciliary motility) can be efficiently assessed in trypanosomes.
ALBA Proteins Are Stage Regulated During Trypanosome Development in the Tsetse Fly and Participate in Differentiation Molecular Biology of the Cell. Nov, 2011 | Pubmed ID: 21965287 The protozoan parasite Trypanosoma brucei is responsible for sleeping sickness and alternates between mammal and tsetse fly hosts, where it has to adapt to different environments. We investigated the role of two members of the ALBA family, which encodes hypothetical RNA-binding proteins conserved in most eukaryotes. We show that ALBA3/4 proteins colocalize with the DHH1 RNA-binding protein and with a subset of poly(A+) RNA in stress granules upon starvation. Depletion of ALBA3/4 proteins by RNA interference in the cultured procyclic stage produces cell modifications mimicking several morphogenetic aspects of trypanosome differentiation that usually take place in the fly midgut. A combination of immunofluorescence data and videomicroscopy analysis of live trypanosomes expressing endogenously ALBA fused with fluorescent proteins revealed that ALBA3/4 are present throughout the development of the parasite in the tsetse fly, with the striking exception of the transition stages found in the proventriculus region. This involves migration of the nucleus toward the posterior end of the cell, a phenomenon that is perturbed upon forced expression of ALBA3 during the differentiation process, showing for the first time the involvement of an RNA-binding protein in trypanosome development in vivo.
NUP-1 Is a Large Coiled-coil Nucleoskeletal Protein in Trypanosomes with Lamin-like Functions PLoS Biology. 2012 | Pubmed ID: 22479148 A unifying feature of eukaryotic nuclear organization is genome segregation into transcriptionally active euchromatin and transcriptionally repressed heterochromatin. In metazoa, lamin proteins preserve nuclear integrity and higher order heterochromatin organization at the nuclear periphery, but no non-metazoan lamin orthologues have been identified, despite the likely presence of nucleoskeletal elements in many lineages. This suggests a metazoan-specific origin for lamins, and therefore that distinct protein elements must compose the nucleoskeleton in other lineages. The trypanosomatids are highly divergent organisms and possess well-documented but remarkably distinct mechanisms for control of gene expression, including polycistronic transcription and trans-splicing. NUP-1 is a large protein localizing to the nuclear periphery of Trypanosoma brucei and a candidate nucleoskeletal component. We sought to determine if NUP-1 mediates heterochromatin organization and gene regulation at the nuclear periphery by examining the influence of NUP-1 knockdown on morphology, chromatin positioning, and transcription. We demonstrate that NUP-1 is essential and part of a stable network at the inner face of the trypanosome nuclear envelope, since knockdown cells have abnormally shaped nuclei with compromised structural integrity. NUP-1 knockdown also disrupts organization of nuclear pore complexes and chromosomes. Most significantly, we find that NUP-1 is required to maintain the silenced state of developmentally regulated genes at the nuclear periphery; NUP-1 knockdown results in highly specific mis-regulation of telomere-proximal silenced variant surface glycoprotein (VSG) expression sites and procyclin loci, indicating a disruption to normal chromatin organization essential to life-cycle progression. Further, NUP-1 depletion leads to increased VSG switching and therefore appears to have a role in control of antigenic variation. Thus, analogous to vertebrate lamins, NUP-1 is a major component of the nucleoskeleton with key roles in organization of the nuclear periphery, heterochromatin, and epigenetic control of developmentally regulated loci.
A New Asymmetric Division Contributes to the Continuous Production of Infective Trypanosomes in the Tsetse Fly Development (Cambridge, England). May, 2012 | Pubmed ID: 22491946 African trypanosomes are flagellated protozoan parasites that cause sleeping sickness and are transmitted by the bite of the tsetse fly. To complete their life cycle in the insect, trypanosomes reach the salivary glands and transform into the metacyclic infective form. The latter are expelled with the saliva at each blood meal during the whole life of the insect. Here, we reveal a means by which the continuous production of infective parasites could be ensured. Dividing trypanosomes present in the salivary glands of infected tsetse flies were monitored by live video-microscopy and by quantitative immunofluorescence analysis using molecular markers for the cytoskeleton and for surface antigens. This revealed the existence of two distinct modes of trypanosome proliferation occurring simultaneously in the salivary glands. The first cycle produces two equivalent cells that are not competent for infection and are attached to the epithelium. This mode of proliferation is predominant at the early steps of infection, ensuring a rapid colonization of the glands. The second mode is more frequent at later stages of infection and involves an asymmetric division. It produces a daughter cell that matures into the infective metacyclic form that is released in the saliva, as demonstrated by the expression of specific molecular markers - the calflagins. The levels of these calcium-binding proteins increase exclusively in the new flagellum during the asymmetric division, showing the commitment of the future daughter cell to differentiation. The coordination of these two alternative cell cycles contributes to the continuous production of infective parasites, turning the tsetse fly into an efficient and long-lasting vector for African trypanosomes.
Intraflagellar Transport Proteins Cycle Between the Flagellum and Its Base Journal of Cell Science. Jan, 2013 | Pubmed ID: 22992454 Intraflagellar transport (IFT) is necessary for the construction of cilia and flagella. IFT proteins are concentrated at the base of the flagellum but little is known about the actual role of this pool of proteins. Here, IFT was investigated in Trypanosoma brucei, an attractive model for flagellum studies, using GFP fusions with IFT52 or the IFT dynein heavy chain DHC2.1. Tracking analysis by a curvelet method allowing automated separation of forward and return transport demonstrated a uniform speed for retrograde IFT (5 µm s(-1)) but two distinct populations for anterograde movement that are sensitive to temperature. When they reach the distal tip, anterograde trains are split into three and converted to retrograde trains. When a fast anterograde train catches up with a slow one, it is almost twice as likely to fuse with it rather than to overtake it, implying that these trains travel on a restricted set of microtubules. Using photobleaching experiments, we show for the first time that IFT proteins coming back from the flagellum are mixed with those present at the flagellum base and can reiterate a full IFT cycle in the flagellum. This recycling is dependent on flagellum length and IFT velocities. Mathematical modelling integrating all parameters actually reveals the existence of two pools of IFT proteins at the flagellum base, but only one is actively engaged in IFT.
Trypanosoma Brucei FKBP12 Differentially Controls Motility and Cytokinesis in Procyclic and Bloodstream Forms Eukaryotic Cell. Feb, 2013 | Pubmed ID: 23104568 FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton.
Apoptotic Marker Expression in the Absence of Cell Death in Staurosporine-treated Leishmania Donovani Antimicrobial Agents and Chemotherapy. Mar, 2013 | Pubmed ID: 23263009 The protozoan parasite Leishmania donovani undergoes several developmental transitions in its insect and vertebrate hosts that are induced by environmental changes. The roles of protein kinases in these adaptive differentiation steps and their potential as targets for antiparasitic intervention are only poorly characterized. Here, we used the generic protein kinase inhibitor staurosporine to gain insight into how interference with phosphotransferase activities affects the viability, growth, and motility of L. donovani promastigotes in vitro. Unlike the nonkinase drugs miltefosine and amphotericin B, staurosporine strongly reduced parasite biosynthetic activity and had a cytostatic rather than a cytotoxic effect. Despite the induction of a number of classical apoptotic markers, including caspase-like activity and surface binding of annexin V, we determined that, on the basis of cellular integrity, staurosporine did not cause cell death but caused cell cycle arrest and abrogated parasite motility. In contrast, targeted inhibition of the parasite casein kinase 1 (CK1) protein family by use of the CK1-specific inhibitor D4476 resulted in cell death. Thus, pleiotropic inhibition of L. donovani protein kinases and possibly other ATP-binding proteins by staurosporine dissociates apoptotic marker expression from cell death, which underscores the relevance of specific rather than broad kinase inhibitors for antiparasitic drug development.
Molecular Basis of Tubulin Transport Within the Cilium by IFT74 and IFT81 Science (New York, N.Y.). Aug, 2013 | Pubmed ID: 23990561 Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.
Getting to the Heart of Intraflagellar Transport Using Trypanosoma and Chlamydomonas Models: the Strength is in Their Differences Cilia. Nov, 2013 | Pubmed ID: 24289478 Cilia and flagella perform diverse roles in motility and sensory perception, and defects in their construction or their function are responsible for human genetic diseases termed ciliopathies. Cilia and flagella construction relies on intraflagellar transport (IFT), the bi-directional movement of 'trains' composed of protein complexes found between axoneme microtubules and the flagellum membrane. Although extensive information about IFT components and their mode of action were discovered in the green algae Chlamydomonas reinhardtii, other model organisms have revealed further insights about IFT. This is the case of Trypanosoma brucei, a flagellated protist responsible for sleeping sickness that is turning out to be an emerging model for studying IFT. In this article, we review different aspects of IFT, based on studies of Chlamydomonas and Trypanosoma. Data available from both models are examined to ask challenging questions about IFT such as the initiation of flagellum construction, the setting-up of IFT and the mode of formation of IFT trains, and their remodeling at the tip as well as their recycling at the base. Another outstanding question is the individual role played by the multiple IFT proteins. The use of different models, bringing their specific biological and experimental advantages, will be invaluable in order to obtain a global understanding of IFT.
More Than Meets the Eye: Understanding Trypanosoma Brucei Morphology in the Tsetse Frontiers in Cellular and Infection Microbiology. 2013 | Pubmed ID: 24312899 T. brucei, the causative parasite for African trypanosomiasis, faces an interesting dilemma in its life cycle. It has to successfully complete its infection cycle in the tsetse vector to be able to infect other vertebrate hosts. T. brucei has to undergo multiple morphological changes as it invades the alimentary canal of the tsetse to finally achieve infectivity in the salivary glands. In this review, we attempt to elucidate how these morphological changes are possible for a parasite that has evolved a highly robust cell structure to survive the chemically and physically diverse environments it finds itself in. To achieve this, we juxtaposed the experimental evidence that has been collected from T. brucei forms that are cultured in vitro with the observations that have been carried out on tsetse-infective forms in vivo. Although the accumulated knowledge on T. brucei biology is by no means trivial, several outstanding questions remain for how the parasite mechanistically changes its morphology as it traverses the tsetse and how those changes are triggered. However, we conclude that with recent breakthroughs allowing for the replication of the tsetse-infection process of T. brucei in vitro, these outstanding questions can finally be addressed.
Forward Motility is Essential for Trypanosome Infection in the Tsetse Fly Cellular Microbiology. Mar, 2014 | Pubmed ID: 24134537 African trypanosomes are flagellated protozoan parasites transmitted by the bite of tsetse flies and responsible for sleeping sickness in humans. Their complex development in the tsetse digestive tract requires several differentiation and migration steps that are thought to rely on trypanosome motility. We used a functional approach in vivo to demonstrate that motility impairment prevents trypanosomes from developing in their vector. Deletion of the outer dynein arm component DNAI1 results in strong motility defects but cells remain viable in culture. However, although these mutant trypanosomes could infect the tsetse fly midgut, they were neither able to reach the foregut nor able to differentiate into the next stage, thus failing to complete their parasite cycle. This is the first in vivo demonstration that trypanosome motility is essential for the accomplishment of the parasite cycle.
Flagellar Adhesion in Trypanosoma Brucei Relies on Interactions Between Different Skeletal Structures in the Flagellum and Cell Body Journal of Cell Science. Jan, 2014 | Pubmed ID: 24163437 The Trypanosoma brucei flagellum is an essential organelle anchored along the surface of the cell body through a specialized structure called the flagellum attachment zone (FAZ). Adhesion relies on the interaction of the extracellular portion of two transmembrane proteins, FLA1 and FLA1BP. Here, we identify FLAM3 as a novel large protein associated with the flagellum skeleton whose ablation inhibits flagellum attachment. FLAM3 does not contain transmembrane domains and its flagellar localization matches closely, but not exactly, that of the paraflagellar rod, an extra-axonemal structure present in the flagellum. Knockdown of FLA1 or FLAM3 triggers similar defects in motility and morphogenesis, characterized by the assembly of a drastically reduced FAZ filament. FLAM3 remains associated with the flagellum skeleton even in the absence of adhesion or a normal paraflagellar rod. However, the protein is dispersed in the cytoplasm when flagellum formation is inhibited. By contrast, FLA1 remains tightly associated with the FAZ filament even in the absence of a flagellum. In these conditions, the extracellular domain of FLA1 points to the cell surface. FLAM3 is essential for proper distribution of FLA1BP, which is restricted to the most proximal portion of the flagellum upon knockdown of FLAM3. We propose that FLAM3 is a key component of the FAZ connectors that link the axoneme to the adhesion zone, hence it acts in an equivalent manner to the FAZ filament complex, but on the side of the flagellum.
Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma Brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics Molecular & Cellular Proteomics : MCP. Jul, 2014 | Pubmed ID: 24741115 Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.
The Leishmania Donovani Chaperone Cyclophilin 40 is Essential for Intracellular Infection Independent of Its Stage-specific Phosphorylation Status Molecular Microbiology. Jul, 2014 | Pubmed ID: 24811325 During its life cycle, the protozoan pathogen Leishmania donovani is exposed to contrasting environments inside insect vector and vertebrate host, to which the parasite must adapt for extra- and intracellular survival. Combining null mutant analysis with phosphorylation site-specific mutagenesis and functional complementation we genetically tested the requirement of the L. donovani chaperone cyclophilin 40 (LdCyP40) for infection. Targeted replacement of LdCyP40 had no effect on parasite viability, axenic amastigote differentiation, and resistance to various forms of environmental stress in culture, suggesting important functional redundancy to other parasite chaperones. However, ultrastructural analyses and video microscopy of cyp40-/- promastigotes uncovered important defects in cell shape, organization of the subpellicular tubulin network and motility at stationary growth phase. More importantly, cyp40-/- parasites were unable to establish intracellular infection in murine macrophages and were eliminated during the first 24 h post infection. Surprisingly, cyp40-/- infectivity was restored in complemented parasites expressing a CyP40 mutant of the unique S274 phosphorylation site. Together our data reveal non-redundant CyP40 functions in parasite cytoskeletal remodelling relevant for the development of infectious parasites in vitro independent of its phosphorylation status, and provide a framework for the genetic analysis of Leishmania-specific phosphorylation sites and their role in regulating parasite protein function.
The GTPase IFT27 is Involved in Both Anterograde and Retrograde Intraflagellar Transport ELife. Apr, 2014 | Pubmed ID: 24843028 The construction of cilia and flagella depends on intraflagellar transport (IFT), the bidirectional movement of two protein complexes (IFT-A and IFT-B) driven by specific kinesin and dynein motors. IFT-B and kinesin are associated to anterograde transport whereas IFT-A and dynein participate to retrograde transport. Surprisingly, the small GTPase IFT27, a member of the IFT-B complex, turns out to be essential for retrograde cargo transport in Trypanosoma brucei. We reveal that this is due to failure to import both the IFT-A complex and the IFT dynein into the flagellar compartment. To get further molecular insight about the role of IFT27, GDP- or GTP-locked versions were expressed in presence or absence of endogenous IFT27. The GDP-locked version is unable to enter the flagellum and to interact with other IFT-B proteins and its sole expression prevents flagellum formation. These findings demonstrate that a GTPase-competent IFT27 is required for association to the IFT complex and that IFT27 plays a role in the cargo loading of the retrograde transport machinery.DOI: http://dx.doi.org/10.7554/eLife.02419.001.
The Intraflagellar Transport Dynein Complex of Trypanosomes is Made of a Heterodimer of Dynein Heavy Chains and of Light and Intermediate Chains of Distinct Functions Molecular Biology of the Cell. Sep, 2014 | Pubmed ID: 24989795 Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140(RNAi) mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex.
[Elongation of the Axoneme and Dynamics of Intraflagellar Transport] Medecine Sciences : M/S. Nov, 2014 | Pubmed ID: 25388576 Cilia and flagella are essential organelles in most eukaryotes including human beings. In this review, we will discuss the mode of assembly of these complex organelles that depends on a dynamic process called intraflagellar transport or IFT. IFT delivers structural elements at the distal end of the cilium where assembly takes place, thereby allowing the growth of the organelle. We next discuss the different models for control of cilium length and their alterations in ciliopathies, genetic diseases associated to ciliary defects.
Generation of a Nanobody Targeting the Paraflagellar Rod Protein of Trypanosomes PloS One. 2014 | Pubmed ID: 25551637 Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa), costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa), antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392) targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1) of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi) were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392) that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.
Social Motility in African Trypanosomes: Fact or Model? Trends in Parasitology. Feb, 2015 | Pubmed ID: 25577311 African trypanosomes grown on agarose plates exhibit behaviours akin to social motility. This phenomenon has not been observed in vivo so far but recently turned out to be instrumental in the definition of two specific stages of the parasite cycle and as a tool to probe for trypanosome sensing functions.
Imaging Intraflagellar Transport in Trypanosomes Methods in Cell Biology. 2015 | Pubmed ID: 25837405 Trypanosoma brucei is a flagellated eukaryotic pathogen responsible for sleeping sickness in central Africa. Because of the presence of a long motile flagellum (>20 μm) and its amenity to genetic manipulation, it is becoming an attractive model to study the assembly and the functions of cilia and flagella. In recent years, several aspects have been investigated, especially intraflagellar transport (IFT) that has been exhaustively characterized at the light microscopy level. In this manuscript, we review various methods to express fluorescent fusion proteins and to record IFT in living trypanosomes in normal or mutant contexts. We present an approach for separating anterograde and retrograde IFT, hence facilitating quantification of train speed, frequency, and size. A statistical analysis to discriminate different subpopulations of IFT trains is also summarized. These methods have proven their efficiency for the study of IFT in trypanosomes and could be applied to any other organism.
The More We Know, the More We Have to Discover: an Exciting Future for Understanding Cilia and Ciliopathies Cilia. 2015 | Pubmed ID: 25974046 The Cilia 2014 conference was organised by four European networks: the Ciliopathy Alliance, the Groupement de Recherche CIL, the Nordic Cilia and Centrosome Network and the EU FP7 programme SYSCILIA. More than 400 delegates from 27 countries gathered at the Institut Pasteur conference centre in Paris, including 30 patients and patient representatives. The meeting offered a unique opportunity for exchange between different scientific and medical communities. Major highlights included new discoveries about the roles of motile and immotile cilia during development and homeostasis, the mechanism of cilium construction, as well as progress in diagnosis and possible treatment of ciliopathies. The contributions to the cilia field of flagellated infectious eukaryotes and of systems biology were also presented.
Scanning Transmission Electron Microscopy Through-focal Tilt-series on Biological Specimens Micron (Oxford, England : 1993). Oct, 2015 | Pubmed ID: 26093182 Since scanning transmission electron microscopy can produce high signal-to-noise ratio bright-field images of thick (≥500 nm) specimens, this tool is emerging as the method of choice to study thick biological samples via tomographic approaches. However, in a convergent-beam configuration, the depth of field is limited because only a thin portion of the specimen (from a few nanometres to tens of nanometres depending on the convergence angle) can be imaged in focus. A method known as through-focal imaging enables recovery of the full depth of information by combining images acquired at different levels of focus. In this work, we compare tomographic reconstruction with the through-focal tilt-series approach (a multifocal series of images per tilt angle) with reconstruction with the classic tilt-series acquisition scheme (one single-focus image per tilt angle). We visualised the base of the flagellum in the protist Trypanosoma brucei via an acquisition and image-processing method tailored to obtain quantitative and qualitative descriptors of reconstruction volumes. Reconstructions using through-focal imaging contained more contrast and more details for thick (≥500 nm) biological samples.
The Flagellar Arginine Kinase in Trypanosoma Brucei Is Important for Infection in Tsetse Flies PloS One. 2015 | Pubmed ID: 26218532 African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK) including one (AK3) that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands) as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins.
IFT81, Encoding an IFT-B Core Protein, As a Very Rare Cause of a Ciliopathy Phenotype Journal of Medical Genetics. Oct, 2015 | Pubmed ID: 26275418 Bidirectional intraflagellar transport (IFT) consists of two major protein complexes, IFT-A and IFT-B. In contrast to the IFT-B complex, all components of IFT-A have recently been linked to human ciliopathies when defective. We therefore hypothesised that mutations in additional IFT-B encoding genes can be found in patients with multisystemic ciliopathies.
Flagellar Membranes Are Rich in Raft-forming Phospholipids Biology Open. Aug, 2015 | Pubmed ID: 26276100 The observation that the membranes of flagella are enriched in sterols and sphingolipids has led to the hypothesis that flagella might be enriched in raft-forming lipids. However, a detailed lipidomic analysis of flagellar membranes is not available. Novel protocols to detach and isolate intact flagella from Trypanosoma brucei procyclic forms in combination with reverse-phase liquid chromatography high-resolution tandem mass spectrometry allowed us to determine the phospholipid composition of flagellar membranes relative to whole cells. Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells. In contrast, phosphatidylcholine and phosphatidylinositol are strongly depleted in flagella. Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella. Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei.
Intraflagellar Transport is Required for the Maintenance of the Trypanosome Flagellum Composition but Not Its Length Journal of Cell Science. Aug, 2016 | Pubmed ID: 27343245 Intraflagellar transport (IFT) is required for construction of most cilia and flagella. Here, we used electron microscopy, immunofluorescence and live video microscopy to show that IFT is absent or arrested in the mature flagellum of Trypanosoma brucei upon RNA interference (RNAi)-mediated knockdown of IFT88 and IFT140, respectively. Flagella assembled prior to RNAi did not shorten, showing that IFT is not essential for the maintenance of flagella length. Although the ultrastructure of the axoneme was not visibly affected, flagellar beating was strongly reduced and the distribution of several flagellar components was drastically modified. The R subunit of the protein kinase A was no longer concentrated in the flagellum but was largely found in the cell body whereas the kinesin 9B motor was accumulating at the distal tip of the flagellum. In contrast, the distal tip protein FLAM8 was dispersed along the flagellum. This reveals that IFT also functions in maintaining the distribution of some flagellar proteins after construction of the organelle is completed.


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Who knew that the humble spoon had more uses than a utensil merely a device to help stuff our faces with food. The shape of the spoon actually works perfectly in beauty to assist in the application of makeup! Check out some of its awesome uses below!
Isn't it one of the most frustrating things to have done all that blending and your eyeshadow is perfection, then you get mascara smudged on it?!
Here's a lifesaving trick, press the spoon against the eyelid just above the lash line, then apply mascara.
You can do this to the bottom lash line too.
Winged Eyeliner
The perfect wing is hard to achieve on some days, so read one to see how you can always get it right 🙂
Align the spoon handle to follow the natural curves of the bottom lash line.
Then take your eyeliner and just extend the line from the outer corners, following the spoon.
Then take the rounded part of the spoon to connect the line back to the eye to create the perfect cat-eye flick.
Apply to both sides, and done!
Who said eyeliner was hard, right? 🙂
Reduce Puffy Eyes
Put your spoon in the fridge for 10-15 minutes, then hold the curved, underside of the cold spoon under your eyes for about 1 minute. Replace the now warmed spoon with another cold spoon if eyes are still puffy and repeat.
The cold temperature of the spoon helps to tighten up the skin around your eyes and relax the blood vessels, therefore reducing the puffiness surrounding the eyes and on the eye bags. It will also help with brightening the under eye.
Check out our full video below for more beauty hacks using the spoon!
So there you go, you're all ready! 😉
Follow us for more updates: Beauty Within


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Contacting the IT Helpdesk (For current employees)
Direct Phone- (260) 356-0501
Employee Self Service (HR/Payroll)
Click here to log in to Employee Self Service
Click here for Employee Self Service login instructions (PDF)
Remote Access
For Pathfinder Services employees to access their Pathfinder applications, such as email via Citrix, follow these 3 steps (If you have already installed the Citrix software, skip Steps 1 & 2)
If you have already installed the Citrix software and can no longer access Citrix, please click here for instructions (PDF) and if that does not work, call the IT help desk.
Step 1
Download Citrix Receiver: Click here to Download Citrix (PDF)
Step 2
Add https://citrix.pathfinderservices.org to your trusted sites list. Click Here for Instructions (PDF)
(you can print them for reference)
Step 3
Click HERE to Log-in to Citrix
Once you are at the login page, add that page to your Favorites or Bookmarks and you can go right to the login page in the future and skip this page.
In order to gain access to our remote access system you will need the following:
- Citrix Receiver Client (install in Step #1 above)
- Internet Explorer (versions 8 or higher).
In theory, the latest versions of Firefox and Chrome browser should also work. However, these platforms have not been tested and may require additional steps to properly launch the applications.


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Acquire Customers With Exclusive Offers
SheerID's Digital Verification platform instantly verifies your target customers, mitigating fraud while reducing friction in user experience.
Verify credentials in real-time with hundreds of authoritative data sources. Supplement instant verification with full service, fast document review capabilities to verify 100% of the users.
Embed verification within your brand experience with the SheerID API or hosted implementation to create a transparent user experience and effective user acquisition flow.
Secure against abuse with fraud mitigation rules management. Ensure customer data is not shared and is protected with encryption and privacy controls. PCI and SOC2 compliant platform.
The only verification platform that supports a wide range of enterprise use cases: consumer segments (e.g.,military, students), business credentials (e.g., small business), affiliations and more.
30M+ active US service members, veterans, reservists, National Guard, and dependents.
21M currently enrolled students in the US, plus students in over 30 countries worldwide.
6.7M educators in the US, plus faculty in over 30 countries worldwide.
2.7M firefighters, police officers, and EMT's in the US.
44,000 employers with 65M employees in the US.
2.8M US government employees.
Brand ambassadors, influencers, VIP's, and ProDeal program participants
Membership groups, professional associations, civic organizations.
You tell us who is eligible for your exclusive offer.
"Implementation was the smoothest we've had with a third party vendor; we highly recommend SheerID to other Fastspring users looking for verification services."
"If I'm a student applying for a discount, then I am a shopper who is ready to make a purchase. We suspected that if we could speed up the approval process, EDU Advantage members would make their first purchase sooner. Not only did initial purchases occur 50-80 days sooner, students verified by SheerID are spending a little more."
"Scalability and accuracy are the two main reasons we chose SheerID's academic verification solution. SheerID quickly determines who is eligible to receive free access to Tableau's software through our Academic Programs and who isn't."
The best way to understand SheerID's service is to see it in action. Get an overview of the customizable options that are available for your brand and industry. Our sales team is standing by to set up a quick, 15-minute live demo just for you.
Get in touch and let's have a talk about your offers and our solutions.


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Most Pennsylvania parents who are ending their marriages understand that they must put their young children first. However, this is often easier said than done as parents' egos can intervene. If the parents can work together, however, the kids are more likely to be happy and well-adjusted.
For the best results, parents should have a plan that will allow the children to adjust. In many cases, the parenting agreement allows both parents to effectively co-parent. Further, parents who are able to work together can foster similar rules at home so that the kids always know what to expect. By putting the agreement down in writing, the parents will have a road map that they can follow when disputes arise.
The parenting agreement should include certain things to make co-parenting easier for the parents. For example, the plan should outline where the children primarily live and the visitation schedule. The rules and how the children will be disciplined should also be included. The plan may also outline who can see the kids outside of the parents, including grandparents, uncles and aunts. Parents may even come to an agreement on when the kids can meet a new significant other.
Child custody disputes can always create stress for both the parents and the children involved. If parents absolutely cannot come to an agreement, a family law attorney may assist with negotiating a resolution. For example, if the parents cannot agree where the children will attend school, the attorney may provide arguments to the court for one school over the other. If a parent needs to modify their visitation schedule due to a new job or because the children are older and want to be involved in after school activities, the attorney may petition to modify the parenting agreement so that it reflects the parent's new schedule.


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All The Tints Of Friendship Between Women: 10 Nuances One Should Think About
If it turns out that you have too many friends (few friends) worth a think: is it right, do you communicate with everyone well enough, is there a habit to communicate or a force to keep communicating? These nuances will help to find one`s bearings.
1. You do not have to be friends with her just because you are class mates
You we sharing the same desk for two years and were considered to be friends and now she acts strange and say foul things? You are better off without her.
2. You have fun together but it is rather difficult to be friends with her
Yes, she is interesting but she is always late and this is no fun.
3. Co-workers oftentimes are considered to be friends
At least because you spend much time together, communicate, have dinner and know a lot about one another. It is a good thing.
4. If you complain about her – do not be friends with her
No one like it when friends always complain about someone. It will be better to stop it let alone communicate with her.
5. It is better to have few friends but close ones
The best joke is when you call someone a friend but can hardly remember name.
6. Do not be friends forcibly
She likes you, she wants be friends with you, are you? As you wish! It is not necessary.
7. Good ones will remain only
You remember the day you met and always glad to talk? And a good job too!
8. Far from every friend will become the best friend
It is nice to watch a movie with some friends, to go to a bar with others and some are good to cry over a shoulder and after that what a jerk this ex is. Jump to conclusions on your own.
9. It is strange to have not a single friend
Perhaps, it is the way society influences that people stopped knowing a thing about each other, but at least one friend of the same sex should be over there. Whom to exchange perfumes with, for example?
10. One may always become better
Those are sandwiches at night and taking care about one another and some other small potatoes, which do not cost anything but are highly appreciated.
About the author:
Melisa Marzett is one-of-a-kind author, writer and blogger who enjoys the process of writing. Working for http://star-writers.com/ currently, she enjoys every single day she spends writing because this is what she is fond of and she can`t imagine a single day without writing.
Akshay Sharma
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Book your place on our 2018 Open Days
Our open days are a chance to learn more about our courses, meet staff and students, see academic facilities, view accommodation and explore our campus and the city.
This year our open days will be held on:
- Friday 15 June
- Saturday 16 June
- Saturday 8 September
- Saturday 6 October
But don't worry, if you can't make an open day, our campus tours are available throughout most of the year.
Applicant Open Days
When we receive your UCAS form we will write with details of our offer and invite you to visit and perhaps have an interview (dependent on programme). You can use our web-based booking system to reserve a place on a suitable date, and let us know if you want to bring a parent or friend with you.


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It's natural to want to look good and healthy for the summer, but how?
Dr Harker can relax your frown lines and 'crow's feet' for you to improve the lines from nose to mouth, whilst also helping the 'bar code lines above the lips and the "sad lines" from the corners of the mouth and also subtly augment the lips to be fuller and youthful again.
You may have some life-long moles that you hate or perhaps skin tags & blemishes that you'd like to get rid of before your holiday, so with Dr Harker at The Erme Clinic there is no need to put up with unsightly moles any more, you know the ones… the ones that people notice before they notice you!
In dermatology we can label moles "the good, the bad and the ugly", but unfortunately, most moles fall in the 'ugly' category. However there could be a solution! Dr Harker will carefully check, before quickly, simply & safely removing the mole for you. Dr Harker will then also arrange for the mole to be checked under the microscope to ensure there is nothing sinister going on for your peace of mind.
We realise now that most of us have far more UV exposure than in our grandparents' day and this is due to the depletion of the ozone layer, longer leisure hours and foreign holidays. UV damage accumulates over time, so we need to be sensible and protect our skin as much as possible. Our advice is for you to slip on a t-shirt, slap on some sun lotion and wear a broad brimmed hat, all of which can help considerably!
Fair skin, combined with fair or red hair, is the most at risk, however it isn't just limited to this as all skin is at risk! Imagine hanging a lovely soft velvety chamois leather out on the line in April and coming back to see what it'll look like when you take it down in October?! It will be hard, brittle and cracked and resemble something like crocodile skin….not attractive!
One way to improve your skin prior to the summer is to have a 'peel' carried out by Dr Harker, as this can improve skin texture whether it is greasy, open pores, dull, wrinkled or discoloured. With a course of 4 or 5 peels this can be improved further and will help refresh the skin and restore a smoother, softer skin with a healthy glow.
The more skin layers peeled off the longer the down-time afterwards, however people have busy lives and can't ill afford extensive time out, this is why most of the peels Dr Harker uses are the superficial ones with a fast recovery time (often a milk peel), this is of course unless the needs of the individual indicate that a medium depth peel is required.
Having improved the skin then it must be protected from further sun damage, so please make sure you take note of our previous hints & tips.
Dr Harker works as a local family doctor and also as a hospital practitioner in Dermatology.She also is on the Board of the British College of Aesthetic Medicine.This work takes her to London and keeps her at the forefront of bringing Aesthetic Medicine under regulatory control.
Dr Ruth Harker is able to offer professional, completely confidential advice for all your annoying little skin problems at her private medical clinic, conveniently based in Ivybridge.
Contact us at the clinic if you are unhappy with any aspect of your skins appearance and Dr Harker will be happy to answer and questions you may have
All work is compliant with NICE guidelines.
It is our policy to refer any problems that can or should be treated on the NHS back to your GP.
Dr Harker offers all these treatments personally.
Details on this website are provided for information purposes only. Opinions voiced reflect Dr Harker's current practice which is subject to change at anytime.
© 2014 - 2018 Erme Clinic|Terms & Conditions|Privacy & Cookie Policy


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Ever seen one of those sci-fi flicks where drones are whizzing around capturing images left, right and center? Have you wondered what it might feel like operating one of them? Well, if you have, you're in luck. This age of science has brought us many gifts, and one of those is that capturing images by drones no longer has to be a figment of your imagination, but a genuine, accessible, possibility.
The drone manufacturing business for the past few years has been booming, so much so that DJI, a photographic drone maker based in China, reports that sales have grown 5 times year on year since 2009. Unsurprising, when you consider the wide market to which it appeals. These drones, dubbed 'flying cameras', are useful for the average person looking for a hobby, or even large institutions like the BBC and Google.
Particularly, they are extremely handy if you want to make a wedding video, as they shoot from angles that are virtually inaccessible for a man with a video camera in his hand. Michael Perry, a spokesman from DJI, describes this as 'a really novel way to get a shot of the entire wedding ceremony'. Other users include farmers, firefighters and just normal people that want to be able to make some really cool videos wherever they go. Developers are working to simplify the use of these devices so that more focus can be put toward the quality of photography than navigation.
However, drones are classified by the Civil Aviation Authority (CAA) as 'unmanned aircraft', so there are certain rules that apply for those of you interested in this. Permission is required from the CAA for the operation of these drones, unless you use them a certain distance away from other people or structures 'not under the pilot's control'. Flight is restricted to 400 feet above surface level in normal circumstances.
If you are planning to use them publicly, CAA tests have to be passed and registration fees have to be paid before clearance can be granted. Admittedly, the dos and don'ts of this matter have proved to be troublesome, the CAA having said that it is 'down to people's common sense'. Another nagging issue with drone usage is privacy. As it is often difficult to determine if the content of drone photography can be classified as journalism or art, whether or not it can be exempted from the Data Protection Act is unclear.
One thing although, is definitely certain. Photographic drones are certainly one of the most exciting prospects in the technological future.


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After sharing a long family history shooting with Olympus equipment, I am now proud to shoot with and be affiliated with Olympus as an official Ambassador for the brand. I am a great believer that "The best camera is the one that you want to pick up and shoot with", and that is why I shoot with Olympus equipment.
Olympus Ambassador events are a great way for all levels to quickly learn from Pro Olympus photographers, so be sure to check out the upcoming events from the link below.


text: The Black Business Student Association (BBSA) brings together past, present, and prospective African-American Darden MBAs and enriches the experience of the broader Darden community by increasing African-American cultural awareness. Our programming focuses on professional development, academic excellence, social activities, community service, and safe spaces for the Darden community to discuss race related topics. Membership is open to all.Members of BBSA recently hosted a webinar where they shared their experiences at Darden and what their club brings to the Darden community. Although this organization has members who are University of Virginia students and may have University employees associated or engaged in its activities and affairs, the organization is not a part of or an agency of the University. It is a separate and independent organization which is responsible for and manages its own activities and affairs. The University does not direct, supervise or control the organization and is not responsible for the organization's contracts, acts or omissions.Annual EventsThe Black Business Student Association hosts a variety of events and activities throughout the year. Academic Review Sessions BBSA Conference Big Brothers Big Sisters Breast Cancer Awareness Fundraiser Leadership Speaker Series Welcome Back Event with Darden FacultyLeadership TeamWe welcome prospective students to reach out to members of our leadership team to learn more about BBSA. President: Zhana Edmonds Executive Vice President: Marlon Evans Vice President of Corporate Sponsorship: Allison Shimamoto Vice President of Careers: Jordan Clark VP of Finance: Aaron Harrill Vice President of External Relations: Cecily Sackey VP of Marketing and Communications: Christina Gonsalves Contact Us email@example.com